The human APOBEC3G (A3G) protein is a DNA deaminase that has potent activity against HIV-1 and a variety of other retroelements. Although HIV encodes a natural inhibitor of A3G called Vif, we hypothesize that the virus still benefits from the mutagenic activity of A3G (immune escape and drug resistance). As an essential step toward testing this hypothesis in vivo, we have adapted, optimized and miniaturized a fluorescence-based DNA cytidine deaminase assay to high throughput format. Preliminary studies have identified 2 candidate inhibitors in the 1280 compound LOPAC library. Here, we propose two specific aims. First, we will collaborate with an NIH Molecular Libraries Probe Production Center in a full high-throughput screen for additional A3G activity inhibitors. Second, we will implement a variety of secondary screens to determine the specificity of the lead actives. We anticipate that the successful completion of the proposed studies will result in the identification and validation of approximately 50 active DNA deaminase inhibitors. A subset of these small molecules will undoubtedly become tools for many future experiments and further development. PUBLIC HEALTH RELEVANCE: The human protein APOBEC3G has become the prototypic DNA cytidine deaminase. The fact that it can mutate HIV cDNA raises the possibility that modulating its activity may have therapeutic merit.